Synapses are dynamically regulated on the time scale of milliseconds to minutes by the interaction of many forms of plasticity. Such dynamic regulation is poorly understood, even though it plays many crucial roles within the brain and has been implicated in numerous neurological disorders. Our primary goals are to determine the mechanisms and functional consequences of short-term plasticity. By studying multiple forms of plasticity at different types of synapses we will be able to discern general features and synapse-specific specializations tailored to specific functional roles. (1) We will begin by studying posttetanic potentiation (PTP), a widely observed form of use-dependent synaptic enhancement lasting for tens of seconds following high-frequency activation. It had long been thought that sustained presynaptic calcium (Ca) increases contribute to PTP, but the molecular basis of PTP is unclear. We have recently shown that at the calyx of Held PTP is mediated by Ca-dependent protein kinase C. We will now test the hypothesis that PKC senses Ca and produces PTP by phosphorylating Munc18-1, and determine how presynaptic Ca and PKC activation control the time course of PTP. (2) We will also study synaptic regulation that arises from modulation of presynaptic Ca channels. A hallmark of synaptic transmission is that small changes in Ca influx produce large changes in release. This has been thought to arise entirely from alterations in the probability of release and reflect the Ca dependence of synaptotagmin. Remarkably, we find that changes in the effective pool size make large contributions to the Ca dependence of release. We will test the hypothesis that neuromodulators that reduce Ca influx also regulate release in part by decreasing the effective vesicle pool size. (3) Synaptic enhancement can arise by either activating presynaptic ionotropic receptors or subthreshold somatic depolarization. These forms of plasticity involve small depolarizations of presynaptic boutons, but their molecular mechanisms are not known. We will test the hypothesis that they are mediated by a common mechanism: depolarization elevates presynaptic Ca, which activates PKC to enhance release. (4) We will also use optogenetics to overcome current limitations in the study of short-term plasticity. Virtually nothing is known about many important synapses because they cannot be selectively activated with extracellular stimulation in brain slice. Optogenetic tools allow these synapses to be activated, but desensitization and slow kinetics could limit this approach. We will critically evaluate the ability of optogenetic tools to repetitiely activate axons reliably. When appropriate conditions are found, optogenetics will be used to study aspects of short-term plasticity that can't be studied with conventional approaches. Together, these studies will provide new insights into short-term plasticity, and will contribute t the application of new approaches to studying short-term plasticity. PUBLIC HEALTH RELEVANCE: These studies will provide insight into the mechanisms and functional consequences of short-term synaptic plasticity, which dynamically regulate the strength of every synapse in the brain. They will lead to a deeper understanding of how synapses perform computations, and these studies are directly relevant to numerous neurological disorders that involve proteins implicated in short-term plasticity, such as Parkinson's disease, epilepsy and schizophrenia.